The requirement for the amino acid co-germinant during C. difficile spore germination is influenced by mutations in yabG and cspA

Abstract

Clostridium difficile spore germination is critical for the transmission of disease. C. difficile spores germinate in response to cholic acid derivatives, such as taurocholate (TA), and amino acids, such as glycine or alanine. Although the bile acid germinant receptor is known, the amino acid germinant receptor has remained elusive. Here, we used EMS mutagenesis to generate mutants with altered requirements for the amino acid co-germinant, similar to the strategy used previously to identify the bile acid receptor, CspC. Surprisingly, we identified strains that do not require amino acids as co-germinants, and the mutant spores germinated in response to TA alone. Upon sequencing these mutants, we identified different mutations in yabG. In C. difficile, yabG expression is required for the processing of CspBA to CspB and CspA and preproSleC to proSleC during spore formation. A defined yabG mutant exacerbated the EMS mutant phenotype. Moreover, we found that various mutations in cspA caused spores to germinate in the presence of TA alone without the requirement of an amino acid. Thus, our study provides evidence that apart from regulating the CspC levels in the spore, CspA is important for recognition of amino acids as co-germinants during C. difficile spore germination and that two pseudoproteases (CspC and CspA) function as the C. difficile germinant receptors.